.gamma.-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter of the mammalian central nervous system. The rate-limiting step in GABA biosynthesis is the decarboxylation of L-glutamic acid by glutamic acid decarboxylase (GAD). Little is known with certainty regarding the regulation of GAD activity or the expression of GAD genes. Despite its wide distribution in the brain, GAD protein is present in very small quantities and is very difficult to purify to homogeneity.
Studies of GAD have been hindered by the existence of multiple forms of the enzyme, which differ in molecular weight, kinetic properties, sequence (when known), and hydrophobic properties. For example, the presence of three different forms of GAD in porcine brain has been reported (Spink et al., J. Neurochem. 40:1113-1119 (1983)), as well as four forms in rat brain (Spink et al., Brain Res. 421:235-244 (1987)). A mouse brain GAD (Huang et al., Proc. Natl. Acad. Sci. USA 87:8491-8495 (1990)) and a GAD clone isolated from feline brain Kobayashi et al., J. Neurosci. 7:2768-2772 (1987)) have also been reported. At least two isomers of GAD have been reported in human brain. Chang and Gottlieb, J. Neurosci. 8:2123-2130 (1988).
Further complicating the characterization of distinct GAD isozymes is the fact that GADs are also found in tissues outside of the brain, which GADs have varying degrees of homology with brain GADS. For example, GADs are also expressed in germ cells of the testis, as has been reported for several different species, e.g., rat (Persson et al., in Perspectives of Andrology, M. Serio, (ed.), p. 129-138, Raven Press, NY (1989)), and in human, guinea pig, monkey, and mouse testis (Perrson et al., Mol. Cell. Biol. 10:4701-4711 (1990). The human testis GAD was shown to have a relatively high degree of overall nucleotide sequence homology to the feline brain GAD. The presence of GAD in the pancreas has also been described. Okada et al., Science 194:620-622 (1976), Garry et al, J. Histochem. Cytochem. 36:573-580 (1988), and Vincent et al., Neuroendocrin. 36:197-204 (1983).
A rare neurological disorder, termed Stiff-Man Syndrome (SMS), is associated with the presence of autoantibodies to GABA-secreting neurons. The predominant autoantigen for the autoantibodies in SMS has recently been shown to be a brain GAD. Solimena et al., N. Engl. J. Med. 318:1012-1020 (1988) and Solimena et al., N. Engl. J. Med. 322:1555-1560 (1990).
A small proportion of patients with insulin-dependent diabetes mellitus (IDDM) also develop SMS. It has been speculated that autoimmune mechanisms may precipitate the clinical onset of IDDM. The destruction of pancreatic .beta. (beta)-cells in the islets of Langerhans typically precedes IDDM. This destruction is believed to be mediated by a massive infiltration by lymphocytes into the islets, and by the presence of circulating autoantibodies to the .beta.-cells.
A major target of autoantibodies associated with the development of IDDM is a pancreatic .beta.-cell antigen of relative molecular mass 64,000 (64K). Baekkeskov et al., J. Clin. Invest. 79:926-934 (1987). Antibodies to the 64K antigen are present in greater than about 80% of newly diagnosed patients and have been detected up to several years before clinical onset of IDDM, concomitant with a gradual loss of .beta.-cells. The 64K antigen has recently been identified as GAD. Baekkeskov et al., Nature 347:151-156 (1990).
As with brain GADs, the purification of native human islet cell GAD has a number of disadvantages. Human islet GAD is only a trace protein and it would be impractical to isolate useful quantities from natural sources. For example, purification of native human islet cell GAD would require a large number of human pancreata, which itself poses a substantial obstacle. Further, purification from human tissues carries the risk of co-purifying infective agents such as the hepatitis viruses, retroviruses such as HIV-1 and HIV-2, and other viral agents. Not only does this present the possibility of infecting recipients of therapeutic products with such agents, but raises significant concerns among workers who manufacture and test these products as well as those who will use the products in diagnostic laboratories.
There is a need in the art, therefore, for safe methods of producing relatively large amounts of pure preparations of human islet cell GAD polypeptides. These polypeptides would be useful as, inter alia, therapeutic agents in the treatment of GAD-related diseases, such as IDDM, and in the diagnosis and monitoring of these diseases. Quite remarkably, the present invention fulfills these and other related needs through the use of recombinant DNA technology, thus eliminating the problem of viral contamination and providing commercially feasible quantities of human islet cell GAD polypeptides.